Anti-HER2 antibodies and uses thereof

Abstract

The present invention provides anti-HER2 antibodies and uses thereof, particularly a series of anti-HER2 antibodies, which have high binding affinity to HER2, cross-reactivity with human HER2 and cynomolgus monkey HER2, good thermostability, and excellent anti-tumor activity in various tumors (e.g., neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.).

Description

Detailed Description of the Invention

[0001] Technical Field The present invention relates to the field of antibody drugs, in particular to anti-HER2 antibodies and uses thereof.

[0002] Background technology HER2, also known as ErbB2, belongs to the HER subfamily of the type I receptor tyrosine kinase family, which also includes three other members: HER1 (ErbB1 or EGFR), HER3 (ErbB3), and HER4 (ErbB4). HER subfamily members can form homodimers and heterodimers, and HER2 is the most potent dimerization partner with other ErbB receptors. Activation of HER2 results in receptor phosphorylation, which triggers a cascade of downstream signals via multiple signaling pathways, including MAPK, PI3K / AKT, JAK / STAT, and PKC, which may play an important regulatory role in cell proliferation, differentiation, development, adhesion, and migration. Researchers have found that high HER2 expression is associated with many tumors, including neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, and bladder cancer.

[0003] Currently, trastuzumab and pertuzumab are the main HER2-targeting therapeutic antibodies on the market. Trastuzumab recognizes the HER2 extracellular domain IV, and pertuzumab recognizes the HER2 extracellular domain II heterodimerization site, which significantly improves patient survival. However, researchers have also found that trastuzumab has no therapeutic effect on many patients with tumors that highly express HER2.

[0004] Therefore, there remains a need in the field to develop new anti-HER2 antibodies.

[0005] Contents of the present invention After extensive research, the inventors of the present application conducted screening and obtained a series of anti-HER2 antibodies, which have high binding affinity to HER2, cross-reactivity with human HER2 and cynomolgus monkey HER2, good thermostability, and excellent anti-tumor activity against various tumors (e.g., neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.). Therefore, the present invention provides the following aspects.

[0006] Antibody or antigen-binding fragment thereof In a first aspect, (1) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs): (a) VH CDR1 having the structure represented by GFNIKDTY (SEQ ID NO: 10); (b) a VH CDR2 having the structure represented by IYPTQGYT (SEQ ID NO: 11); (c) a VH CDR3 having the structure represented by SRWGGEGFYAMDY (SEQ ID NO: 12); and / or (2) The following three light chain variable region (VL) complementarity-determining regions (CDRs): (d) a VL CDR1 having the structure represented by X1X2VQX3A (SEQ ID NO: 33); (e) a VL CDR2 having the structure represented by SAS (SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27); (f) VL CDR3 having the structure represented by QQHX4X5TPPT (SEQ ID NO: 34) An antibody or an antigen-binding fragment thereof capable of specifically binding to HER2, comprising: During the ceremony, X1 is selected from the amino acid residues Q and N; X2 is selected from the amino acid residues N, Y, and S; X3 is selected from the amino acid residues G and T; X4 is selected from the amino acid residues Y, F, and S; X5 is selected from the amino acid residues S, M, and T; Antibodies or antigen-binding fragments thereof are provided by the present invention.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises: VH CDR1 set forth in SEQ ID NO: 10, VH CDR2 set forth in SEQ ID NO: 11, and VH CDR3 set forth in SEQ ID NO: 12; a VL CDR1 set forth in any one of SEQ ID NOs: 18, 22, and 26, a VL CDR2 set forth in any one of SEQ ID NOs: 19, 23, and 27, and a VL CDR3 set forth in any one of SEQ ID NOs: 20, 24, and 28; Includes:

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 set forth in SEQ ID NO: 10, a VH CDR2 set forth in SEQ ID NO: 11, and a VH CDR3 set forth in SEQ ID NO: 12, and a VL CDR1 set forth in SEQ ID NO: 18, a VL CDR2 set forth in SEQ ID NO: 19, and a VL CDR3 set forth in SEQ ID NO: 20.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 set forth in SEQ ID NO: 10, a VH CDR2 set forth in SEQ ID NO: 11, and a VH CDR3 set forth in SEQ ID NO: 12, and a VL CDR1 set forth in SEQ ID NO: 22, a VL CDR2 set forth in SEQ ID NO: 23, and a VL CDR3 set forth in SEQ ID NO: 24.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 set forth in SEQ ID NO: 10, a VH CDR2 set forth in SEQ ID NO: 11, and a VH CDR3 set forth in SEQ ID NO: 12, and a VL CDR1 set forth in SEQ ID NO: 26, a VL CDR2 set forth in SEQ ID NO: 27, and a VL CDR3 set forth in SEQ ID NO: 28.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO: 9, or a variant thereof, and a VL having the sequence set forth in any one of SEQ ID NOs: 17, 21, and 25, or a variant thereof; The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) or a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived, preferably the substitutions are conservative substitutions.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:17.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:21.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:25.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0017] In some embodiments, the antibody or antigen-binding fragment thereof has a light chain (eg, kappa or lambda) constant region derived from a human immunoglobulin.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region set forth in SEQ ID NO:29 and / or a light chain constant region set forth in SEQ ID NO:31.

[0019] In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, an Fv, a disulfide-linked Fv, an scFv, a diabody, and a single domain antibody (sdAb).

[0020] The antibody of the present invention can be prepared by various methods known in the art, for example, by gene recombination technology.For example, the DNA molecules encoding the heavy and light chains of the antibody of the present invention can be obtained by chemical synthesis or PCR amplification.The obtained DNA molecules are inserted into an expression vector and then transfected into host cells.The transfected host cells are then cultured under specific conditions to express the antibody of the present invention.

[0021] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, such antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells, or Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Additionally, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture. Those skilled in the art will be aware of other techniques for the preparation of such antigen-binding fragments.

[0022] The CDRs of the present invention are delimited according to the IMGT delimitation website http: / / aligncdr.labshare.cn / aligncdr / abrsa.php.

[0023] Single-Arm Antibodies In a second aspect, (1) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs): (a) VH CDR1 having the structure represented by GFNIKDTY (SEQ ID NO: 10); (b) a VH CDR2 having the structure represented by IYPTQGYT (SEQ ID NO: 11); (c) VH CDR3 having the structure represented by SRWGGEGFYAMDY (SEQ ID NO: 12) a first peptide chain comprising: (2) The following three light chain variable region (VL) complementarity-determining regions (CDRs): (d) a VL CDR1 having the structure represented by X1X2VQX3A (SEQ ID NO: 33); (e) a VL CDR2 having the structure represented by SAS (SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27); (f) VL CDR3 having the structure represented by QQHX4X5TPPT (SEQ ID NO: 34) and a second peptide chain comprising: (3) a third peptide chain capable of forming a dimer with the first peptide chain; 1. A single-arm antibody comprising: During the ceremony, X1 is selected from the amino acid residues Q and N; X2 is selected from the amino acid residues N, Y, and S; X3 is selected from the amino acid residues G and T; X4 is selected from the amino acid residues Y, F, and S; X5 is selected from the amino acid residues S, M, and T; Single-arm antibodies are provided by the present invention.

[0024] In some embodiments, the first peptide chain comprises a VH CDR1 set forth in SEQ ID NO:10, a VH CDR2 set forth in SEQ ID NO:11, and a VH CDR3 set forth in SEQ ID NO:12.

[0025] In some embodiments, the second peptide chain comprises a VL CDR1 set forth in any one of SEQ ID NOs: 18, 22, and 26, a VL CDR2 set forth in any one of SEQ ID NOs: 19, 23, and 27, and a VL CDR3 set forth in any one of SEQ ID NOs: 20, 24, and 28.

[0026] In some embodiments, the second peptide chain comprises a VL CDR1 set forth in SEQ ID NO:18, a VL CDR2 set forth in SEQ ID NO:19, and a VL CDR3 set forth in SEQ ID NO:20.

[0027] In some embodiments, the second peptide chain comprises a VL CDR1 set forth in SEQ ID NO:22, a VL CDR2 set forth in SEQ ID NO:23, and a VL CDR3 set forth in SEQ ID NO:24.

[0028] In some embodiments, the second peptide chain comprises a VL CDR1 set forth in SEQ ID NO:26, a VL CDR2 set forth in SEQ ID NO:27, and a VL CDR3 set forth in SEQ ID NO:28.

[0029] In some embodiments, the first peptide chain comprises a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO: 9, or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) or a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, preferably wherein the substitutions are conservative substitutions.

[0030] In some embodiments, the first peptide chain comprises a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO:9.

[0031] In some embodiments, the second peptide chain comprises a light chain variable region (VL) having a sequence set forth in any one of SEQ ID NOs: 17, 21, and 25, or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, preferably wherein the substitutions are conservative substitutions.

[0032] In some embodiments, the second peptide chain comprises a light chain variable region (VL) having a sequence set forth in any one of SEQ ID NOs: 17, 21, and 25.

[0033] In some embodiments, the second peptide chain further comprises a constant region derived from a human immunoglobulin.

[0034] In some embodiments, the second peptide chain comprises a light chain (eg, kappa or lambda) constant region derived from a human immunoglobulin.

[0035] In some embodiments, the second peptide chain comprises the light chain constant region set forth in SEQ ID NO:31.

[0036] In some embodiments, the first peptide chain further comprises a constant region derived from a human immunoglobulin. In some embodiments, the constant region derived from a human immunoglobulin is a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region has a first modification that promotes dimerization between the first peptide chain and a third peptide chain.

[0037] In some embodiments, the third peptide chain comprises an Fc domain monomer. In some embodiments, the Fc domain monomer is an IgG Fc domain monomer, such as an IgG1, IgG2, IgG3, or IgG4 Fc domain monomer. In some embodiments, the Fc domain monomer has a second modification that promotes dimerization of the third peptide chain with the first peptide chain.

[0038] In some embodiments, one of the first modification and the second modification is a "knob" modification and the other is a "hole" modification, thereby forming a "knob-and-hole" modification that promotes dimerization of the first peptide chain with the third peptide chain.

[0039] In some embodiments, the first modification is a "knob" modification and the second modification is a "hole" modification, thereby forming a "knob-into-hole" modification that promotes dimerization of the first peptide chain with a third peptide chain.

[0040] In some embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:30 and the Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO:32.

[0041] This knob-and-hole technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, such that the protrusion is positioned in the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing small amino acid side chains in the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0042] Isolated nucleic acid molecules In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect, or the heavy chain variable region and / or light chain variable region thereof, or encoding the single-arm antibody of the second aspect, or the heavy chain variable region and / or light chain variable region thereof.

[0043] In some embodiments, an isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention, and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present in the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present in different isolated nucleic acid molecules, the isolated nucleic acid molecule of the invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0044] In some embodiments, an isolated nucleic acid molecule comprises a first nucleotide sequence encoding a first peptide chain or a heavy chain variable region thereof of a single-arm antibody of the invention, a second nucleotide sequence encoding a second peptide chain or a light chain variable region of the single-arm antibody, and a third nucleotide sequence encoding a third peptide chain of the single-arm antibody, wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are present in the same or different isolated nucleic acid molecules. When the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are present in different isolated nucleic acid molecules, the isolated nucleic acid molecule of the invention comprises a first nucleic acid molecule comprising the first nucleotide sequence, a second nucleic acid molecule comprising the second nucleotide sequence, and a third nucleic acid molecule comprising the third nucleotide sequence.

[0045] vector In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect. In some embodiments, the vector is a cloning vector or an expression vector.

[0046] In some embodiments, a vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention, and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present in the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present in different vectors, the vector of the invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0047] In some embodiments, a vector comprises a first nucleotide sequence encoding a first peptide chain or a heavy chain variable region thereof of a single-arm antibody of the invention, a second nucleotide sequence encoding a second peptide chain or a light chain variable region thereof, and a third nucleotide sequence encoding a third peptide chain of the single-arm antibody, wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are present in the same or different vectors. When the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are present in different vectors, the vector of the invention comprises a first vector comprising the first nucleotide sequence, a second vector comprising the second nucleotide sequence, and a third vector comprising the third nucleotide sequence.

[0048] host cell In a fifth aspect, the present invention provides a host cell comprising a nucleic acid molecule according to the third aspect or a vector according to the fourth aspect. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0049] Preparation method In a sixth aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof of the first aspect or a single-arm antibody of the second aspect, the method comprising culturing a host cell of the fifth aspect under conditions that allow expression of the antibody, or antigen-binding fragment thereof, or single-arm antibody, and recovering the antibody, or antigen-binding fragment thereof, or single-arm antibody from the culture of the cultured host cells.

[0050] therapeutic use In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0051] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable fluid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable fluid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0052] In an eighth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof of the first aspect, the single-arm antibody of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect or the host cell of the fifth aspect in the manufacture of a medicament for activating HER2, increasing the activity of immune cells, enhancing an immune response, and / or preventing and / or treating a tumour or infection in a subject.

[0053] In some embodiments, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells.

[0054] In some embodiments, the immune response is a HER2-mediated immune response.

[0055] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0056] In some embodiments, the tumor is selected from the group consisting of neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and melanoma.

[0057] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0058] In some embodiments, the subject is a mammal, for example, a human.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof, or single-arm antibody is used alone or in combination with an additional pharmaceutically active agent.

[0060] In a ninth aspect, the present invention provides a method for enhancing an immune response and / or preventing and / or treating a tumor or infection in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the first aspect, or the single-arm antibody of the second aspect, or the pharmaceutical composition of the seventh aspect.

[0061] In some embodiments, the immune response is a HER2-mediated immune response.

[0062] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0063] In some embodiments, the tumor is selected from the group consisting of neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and melanoma.

[0064] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0065] In some embodiments, the subject is a mammal, for example, a human.

[0066] The antibody or antigen-binding fragment thereof, or single-arm antibody, or pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical art, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibody or antigen-binding fragment thereof, or single-arm antibody, or pharmaceutical composition of the present invention must be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the required amount of the antibody or antigen-binding fragment thereof, or single-arm antibody of the present invention in a suitable solvent, optionally incorporating additional desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Additionally, sterile injectable solutions can be prepared as sterile, lyophilized powders (e.g., by vacuum drying or lyophilization) for ease of storage and use. Such sterile, lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0067] The antibody or antigen-binding fragment thereof, or single-arm antibody of the present invention, or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It should be understood by those skilled in the art that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibody or antigen-binding fragment thereof, or single-arm antibody or pharmaceutical composition of the present invention is administered by intravenous injection or bolus injection.

[0068] Detective Use In a tenth aspect, the present invention provides a conjugate comprising an antibody or antigen-binding fragment thereof of the first aspect or a single-arm antibody of the second aspect, and optionally a detectable label conjugated to the antibody or antigen-binding fragment thereof or single-arm antibody.

[0069] In some embodiments, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium ester compounds, luminol, and derivatives thereof or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins), radionuclides, and biotin.

[0070] In an eleventh aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the first aspect or the single-arm antibody of the second aspect or the conjugate of the tenth aspect.

[0071] In some embodiments, the kit comprises the conjugate of the tenth aspect.

[0072] In some embodiments, the kit comprises the antibody or antigen-binding fragment thereof of the first aspect and a second antibody capable of specifically recognizing the antibody or antigen-binding fragment thereof. In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0073] In some embodiments, the kit comprises the single-arm antibody of the second aspect and a second antibody capable of specifically recognizing the single-arm antibody. In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0074] In a twelfth aspect, the present invention provides a method for detecting the presence or level of HER2 in a sample, comprising using an antibody or antigen-binding fragment thereof of the first aspect, or a single-arm antibody of the second aspect, or a conjugate of the tenth aspect. In certain embodiments, the method is used for therapeutic or diagnostic purposes. In other embodiments, the method is used for non-therapeutic and non-diagnostic purposes.

[0075] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0076] In some embodiments, the method comprises using a conjugate of the tenth aspect.

[0077] In some embodiments, the method comprises using the antibody or antigen-binding fragment thereof of the first aspect, and the method further comprises using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment thereof.

[0078] In some embodiments, the method comprises using the single-armed antibody of the second aspect, and the method further comprises using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the single-armed antibody.

[0079] In certain embodiments, the method comprises: (1) contacting a sample with an antibody of the present invention, or an antigen-binding fragment thereof, or a single-arm antibody; and (2) detecting the formation of an immune complex between the antigen and the antibody, or detecting the amount of the immune complex. The formation of an immune complex indicates the presence of HER2 or a cell expressing HER2.

[0080] In a thirteenth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof of the first aspect, or a single-arm antibody of the second aspect, or a conjugate of the tenth aspect, in the manufacture of a detection reagent for detecting the presence or level of HER2 in a sample.

[0081] In some embodiments, the detection reagent detects the presence or level of HER2 in the sample by the method for detecting the presence or level of HER2 in a sample of the twelfth aspect.

[0082] In some embodiments, the sample is a cell sample (eg, an immune cell) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).

[0083] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the virological, biochemical and immunological experimental operation steps used herein are all common steps widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0084] When the terms "for example," "such as," "eg," "including," "comprising," or variations thereof are used herein, such terms are not considered to be limiting terms but are to be interpreted as meaning "but not being limited to" or "not being limited to."

[0085] Unless otherwise specified herein or clearly contradicted by context, the terms "a," "an," and "the" and similar designations should be construed as encompassing the singular and the plural in the context of describing the invention, particularly in the context of the claims which follow.

[0086] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, and this immunoglobulin-derived molecule binds to the target antigen via at least one antigen-binding site located in its variable region. References to the term "antibody" include not only intact antibodies but also antigen-binding fragments capable of specifically binding to a target antigen, unless the context clearly dictates otherwise. An "intact antibody" is typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) light chains and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. This constant domain is not directly involved in binding of the antibody to the antigen, but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into regions of high variability (called complementarity-determining regions (CDRs)), which are interspersed with more conserved regions called framework regions (FRs). V H and V LEach heavy chain / light chain pair consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen-binding site, respectively. The distribution of amino acids in the various regions or domains can be determined according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0087] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Three CDRs are present in each of the heavy and light chain variable regions, and are designated CDR1, CDR2, and CDR3. The exact boundaries of such CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between various numbering systems is well known to those of skill in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0088] As used herein, the terms "framework region" or "FR" residues refer to amino acid residues in antibody variable regions other than the CDR residues defined above.

[0089] The term "antibody" is not limited to any particular method for antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0090] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology by Domantis), probodies, and polypeptides comprising at least a sufficient portion of an antibody to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0091] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." The "full-length heavy chain" refers to a polypeptide chain consisting of, from N- to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; if the full-length antibody is an IgE isotype, it optionally also contains a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain consisting, from N- to C-terminus, of VH, CH1, HR, CH2, and CH3. The "full-length light chain" is a polypeptide chain consisting, from N- to C-terminus, of a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are linked together by disulfide bonds between the CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention may be derived from a single species, such as a human, or may be chimeric or humanized. The full-length antibodies of the present invention comprise two antigen-binding sites, each formed by a pair of a VH and a VL, which specifically recognize and bind to the same antigen.

[0092] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH domain and the CH1 domain; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341: 544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; and the term "Fab' fragment" refers to a fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments of the F(ab')2 fragment and consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH domain and the CH1 domain).

[0093] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. Six CDRs are generally considered to confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although with a lower affinity than the complete binding site.

[0094] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a GGGGS repeat amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731; Choi et al. (2001), Eur. J. Immunol. 31: 94-106; Hu et al. (1996), Cancer Res. 56: 3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56; and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of an scFv. In certain embodiments of the invention, the scFv may form a di-scFv, which refers to an antibody formed by connecting two or more single scFvs in series. In certain embodiments of the invention, the scFv may form a (scFv)2, which refers to an antibody formed by connecting two or more single scFvs in parallel.

[0095] As used herein, the term "diabody" means that the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0096] As used herein, the term "single domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment that is composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) and retains the ability to specifically bind to the same antigen that a full-length antibody binds. Single domain antibodies are also called nanobodies.

[0097] Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds and / or competes with the full-length antibody for specific binding to antigen.

[0098] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antibody antigen-binding fragments can be screened for specificity in the same manner as intact antibodies.

[0099] As used herein, the term "chimeric antibody" refers to an antibody in which some portions of the light and / or heavy chain are derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass), and another portion of the light and / or heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but in either case still retains binding activity for the target antigen (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0100] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region, including CH2 and CH3. The Fc fragment of an antibody has a wide variety of functions but is not involved in antigen binding. "Effector functions" mediated by the Fc region include, inter alia, Fc receptor binding, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. The Fc region may be of any antibody heavy chain constant region isotype, for example, IgG1, IgG2, IgG3, or IgG4.

[0101] The Fc domain can include both native Fc regions and variant Fc regions. Native Fc regions comprise an amino acid sequence identical to that of an Fc region found in nature. For example, native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Variant Fc regions comprise an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification. In some embodiments, variant Fc regions may have altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to native Fc regions. In some embodiments, variant Fc regions may have modifications that promote dimer formation.

[0102] As used herein, the term "identity" refers to the sequence match between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the first amino acid sequence or nucleic acid sequence to optimally align it with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules at this position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of overlapping identical positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0103] Also, the percent identity between two sequences can be determined by using a mathematical algorithm.A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877.Such an algorithm is incorporated into the NBLAST program and XBLAST program of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0104] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, variants have similar, identical, or improved function relative to the polypeptide or peptide from which they are derived.

[0105] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., the reaction between an antibody and a directed antigen. The strength or affinity of a particular binding interaction is determined by the equilibrium dissociation constant (K D) In the present invention, "K D The term "equilibria" refers to the equilibrium constant for dissociation of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the binding between the antibody and the antigen and the higher the affinity between the antibody and the antigen.

[0106] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the association and dissociation rates of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentrations and actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of k / k is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kO, and kD values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured by Biacore using surface plasmon resonance (SPR). Additionally, the dissociation constant can be measured by bioluminescence interferometry or Kinexa.

[0107] As used herein, a detectable label of the present invention can be any substance that can be detected using fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art, and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium ester compounds, luminol, and derivatives thereof, ruthenium derivatives, such as terpyridine ruthenium), magnetic beads (e.g., Dynabeads®), calorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin modified with the above labels for binding to avidin (e.g., streptavidin).

[0108] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication site. As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell, or human cell.

[0109] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing that amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitution of an amino acid residue with an amino acid residue having a similar side chain, for example, substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0110] The 20 conventional amino acids referred to herein are written according to conventional usage.See, for example, Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably.In addition, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art.For example, alanine can be represented by A or Ala.

[0111] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. This is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, etc. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizer has the meaning commonly understood by those skilled in the art. These can stabilize the desired activity of the active ingredient in the drug and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or degradation products thereof (e.g., lactalbumin hydrolysate), etc.In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable fluid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable fluid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0112] As used herein, the term "prevention" refers to a method carried out to prevent or delay the onset of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method carried out to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include (but are not limited to) the alleviation of symptoms, reduction in the extent of disease, stabilization of the condition (i.e., no longer worsening), delay or slowing of the onset of disease, improvement or alleviation of the condition, and symptomatic relief (whether partial or complete), whether detectable or undetectable. In addition, "treatment" also refers to the prolongation of survival compared to expected survival if not treated.

[0113] As used herein, the term "subject" refers to a mammal, e.g., a human. In certain embodiments, the subject (e.g., a human) is suffering from or at risk of suffering from a tumor, infection, or autoimmune disease.

[0114] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect.For example, an effective amount for preventing a disease (e.g., tumor, infection, or autoimmune disease) refers to an amount sufficient to prevent, stop, or delay the onset of the disease, and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease.Determining such an effective amount is well within the capabilities of those skilled in the art.For example, the effective amount for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, drug administration method, and other treatments used in combination, etc.

[0115] Beneficial Effects of the Present Invention The present invention provides anti-HER2 antibodies that have high binding affinity to HER2, cross-reactivity with human HER2 and cynomolgus monkey HER2, good thermostability, and excellent anti-tumor activity against various tumors (e.g., neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.). [Brief explanation of the drawings]

[0116] [Figure 1] FIG. 1 shows a schematic diagram of the structure of the monoclonal antibody and single-arm antibody of the present invention. [Figure 2A] FIG. 2A shows the binding affinity of the monoclonal antibodies of the present invention to the HER2 protein. [Figure 2B] FIG. 2B shows the binding affinity of the monoclonal antibodies of the present invention to the HER2 protein. [Figure 3] FIG. 3 shows the results of detecting the binding of the monoclonal antibody of the present invention to human HER2 on HER2-overexpressing N87 cells. [Figure 4] FIG. 4 shows the results of detecting the blockade of HER2 signal-dependent cell proliferation by the monoclonal antibody of the present invention on HER2-overexpressing N87 cells. [Figure 5]FIG. 5 shows the HER2-mediated ADCC effect of the monoclonal antibodies of the invention. [Figure 6] 6 shows charge heterogeneity analysis of the monoclonal antibody of the present invention after high temperature treatment, where OH represents the sample before high temperature treatment, HT2W represents the sample after 2 weeks of high temperature treatment, and HT4W represents the sample after 4 weeks of high temperature treatment. [Figure 7] 7 shows the activity of the monoclonal antibody of the present invention after high temperature treatment, where 0H represents the sample before high temperature treatment, HT2W represents the sample after 2 weeks of high temperature treatment, and HT4W represents the sample after 4 weeks of high temperature treatment.

[0117] Although embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

[0118] Sequence information Post-translational modification sites (PTMs) can have a negative impact on the stability and safety of drugs, reducing their efficacy. In the present invention, the trastuzumab sequence was optimized to remove these post-translational modification sites (PTMs), and the heavy and light chain CDR regions of the antibody were subjected to random mutation to construct an antibody library. High-affinity antibodies were then screened out from the antibody library using yeast display technology. In the present invention, the amino acid sequences encoding the antibodies are disclosed in detail, and relevant data on the candidate antibodies are further presented.

[0119] Descriptions of the sequences involved in the present invention are provided in Tables 1-1 and 1-2 below. [Table 1-1] [Table 1-2]

[0120] SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 2 GFNIKDTY SEQ ID NO: 3 IYPTNGYT SEQ ID NO:4 SRWGGDGFYAMDY SEQ ID NO:5 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0121] SEQ ID NO:6 QDVNTA SEQ ID NO:7 SAS SEQ ID NO:8 QQHYTTPPT SEQ ID NO:9 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS SEQ ID NO: 10 GFNIKDTY

[0122] SEQ ID NO: 11 IYPTQGYT SEQ ID NO: 12 SRWGGEGFYAMDY SEQ ID NO: 13 DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID NO: 14 QDVQTA SEQ ID NO: 15 SAS

[0123] SEQ ID NO: 16 QQHYTTPPT SEQ ID NO: 17 DIQMTQSPSSLSASVGDRVTITCRASQNVQGAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYSTPPTFGQGTKVEIK SEQ ID NO: 18 QNVQGA SEQ ID NO: 19 SAS SEQ ID NO: 20 QQHYSTPPT

[0124] SEQ ID NO: 21 DIQMTQSPSSLSASVGDRVTITCRASNYVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHFMTPPTFGQGTKVEIK SEQ ID NO: 22 NYVQTA SEQ ID NO: 23 SAS SEQ ID NO: 24 QQHFMTPPT SEQ ID NO: 25 DIQMTQSPSSLSASVGDRVTITCRASQSVQGAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHSTTPPTFGQGTKVEIK

[0125] SEQ ID NO: 26 QSVQGA SEQ ID NO: 27 SAS SEQ ID NO: 28 QQHSTTPPT SEQ ID NO: 29 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 30 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0126] SEQ ID NO: 31 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [Example]

[0127] Specific Models for Practicing the Invention The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0128] Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention are basically performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989 and F.M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the manufacturers. Those skilled in the art will appreciate that the examples are provided to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0129] Example 1: Construction and screening of a monoclonal antibody affinity maturation library 1.1. Construction of affinity maturation libraries The heavy chain variable region gene fragment (amino acid sequence shown in SEQ ID NO: 1), the light chain variable region gene fragment (amino acid sequence shown in SEQ ID NO: 5), the heavy chain variable region gene fragment with N62Q and D110E mutations (amino acid sequence shown in SEQ ID NO: 9), and the light chain variable region gene fragment with N36Q mutation (amino acid sequence shown in SEQ ID NO: 13) were synthesized. Using PTM-removed VH and VK as templates, degenerate primers were designed for the CDR2 and CDR3 of VH and the CDR3 of VK, respectively. To ensure that each amino acid could be mutated to any of the 20 amino acids, PCR amplification was performed using NNK as the mutagenic base type. The target fragments were recovered using a PCR purification kit (purchased from QIAGEN). The linearized yeast display vector and the VH and VK PCR products were mixed and electrotransformed into Saccharomyces cerevisiae to construct affinity maturation libraries of heavy and light chain mutants, respectively, and the performance of these libraries was determined.

[0130] 1.2. Screening for anti-HER2 antibodies 1.2.1 Biotinylation of HER2 protein An appropriate amount of double-distilled water was collected and human HER2 protein (purchased from AcroBiosystems) was dissolved in. Following the instructions of the biotin labeling kit (purchased from Thermo), biotin was dissolved and mixed with the protein solution, followed by incubation at 4°C for 2 hours. Excess biotin was removed using a desalting column (purchased from Thermo). All pretreatment steps for this desalting column and sample collection were performed according to the instructions of the product.

[0131] 1.2.2 MACS enrichment of yeast cells capable of specifically binding to human HER2 The antibody library constructed in Example 1.1 was inoculated into SD-CAA amplification medium (1 L of SD-CAA amplification medium containing 6.7 g of YNB, 5 g of casamino acids, 13.62 g of NaHPO 12H O, 7.44 g of NaHPO, and 2% glucose) and cultured overnight at 30°C and 225 rpm. An appropriate amount of yeast cells was harvested and centrifuged at 3,000 rpm for 5 minutes (the subsequent centrifugation was the same procedure) to remove the medium. The yeast cells were resuspended in SD-CAA induction medium and induced overnight. After induction, the library concentration was determined. An appropriate amount of yeast cells was harvested and centrifuged to remove the medium. The yeast cells were resuspended in 50 ml of PBS and centrifuged to remove the supernatant. The yeast cells were resuspended in 10 ml of PBS.

[0132] Biotin-labeled human HER2 protein (final concentration 100 nM) was added and incubated at room temperature for 30 minutes. Yeast cells were collected by centrifugation and washed three times with 50 mL of PBS. The yeast cells were resuspended in 5 mL of wash solution, and 200 μL of SA magnetic beads (purchased from Miltenyi Biotec) was added. The mixture was then inverted and incubated for 10 minutes. The yeast cell and magnetic bead mixture was washed three times with PBS, and then the mixture was applied to an LS purification column (purchased from Miltenyi Biotec). The LS purification column was placed on a magnetic grid, and nonspecifically bound yeast cells were removed by washing with PBS. The purification column was removed from the magnetic grid, and the yeast cells were eluted by adding PBS. The eluted yeast cells were centrifuged and then transferred to SD-CAA amplification medium for amplification.

[0133] 1.2.3 Flow cytometric selection for high affinity yeast cells Yeast cells enriched by MACS were seeded in SD-CAA amplification medium and grown overnight at 30°C and 225 rpm in a shaker flask. The yeast cells were resuspended in SD-CAA induction medium and induced overnight. Anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled HER2 antigen were added and incubated for 10 minutes. The yeast cells were washed three times with PBS, and then goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin-APC conjugated fluorescent antibody (purchased from Invitrogen) were added and incubated for 15 minutes. The cells were resuspended in PBS and sorted using a BD FACSAria III instrument to identify yeast cells with high binding affinity to the HER2 antigen.

[0134] 1.2.4 Collection of antibody genes for HER2 antibody candidate molecules Yeast solutions with high binding ability to human HER2 antigen obtained by MACS and FACS enrichment were coated onto SD-CAA solid culture plates, and single clones were selected and cultured overnight at 30°C and 225 rpm in SD-CAA amplification medium. The amplified single clones were treated with 0.1% SDS, centrifuged, and subjected to PCR amplification using the supernatant as a template. The PCR products were sequenced to obtain the gene sequences.

[0135] Example 2: Construction, expression, and purification of monoclonal antibodies 2.1. Construction of monoclonal antibody genes in the pCDNA3.1 expression vector The heavy chain variable region gene sequence was ligated to the human IgG1 constant region gene sequence (amino acid sequence set forth in SEQ ID NO:29) and constructed into an EcoR I / Not I double-digested linearized pCDNA3.1 vector using homologous recombinase (purchased from Vazyme). The light chain variable region gene sequence was ligated to the human kappa constant region gene sequence (amino acid sequence set forth in SEQ ID NO:31) and constructed into an EcoR I / Xhol I double-digested linearized pCDNA3.1 vector. This process was performed according to the manufacturer's instructions. The homologous recombination product was transferred into Top10 competent cells, which were plated on ampicillin-resistant plates and cultured overnight at 37°C. Single clones were selected and sequenced, and plasmids were extracted.

[0136] 2.2. Cell transfection and protein purification HEK293 cells were used for transfection and expression. The cell density was adjusted to 2.5 × 10 cells one day before transfection. 6 The next day, the cells were diluted to 3.0 × 10 6 Transfection was performed by diluting the solution to 1000 cells / ml. MEM medium was used as the transfection buffer. PEI was added at a PEI:plasmid ratio of 3:1. The extracted heavy and light chain plasmids were co-transfected into HEK293 cells. Five days after cell culture, the supernatant was collected and the target protein was purified using a Protein A-loaded column. Ten column volumes of PBS were added to equilibrate the loaded column, and the cell supernatant was loaded onto the gravity column and allowed to flow through under gravity. After loading the sample, 20 column volumes of PBS were added to wash away unbound impurities. When no more liquid flowed out, the column was placed in a collection tube pre-filled with neutralization buffer (1 M Tris, pH 8.54), and the target protein was eluted using 3–5 column volumes of elution buffer (0.1 M sodium citrate, pH 3.2).

[0137] Example 3: Construction, expression, and purification of anti-HER2 single-arm antibodies To further confirm the affinity of the monovalent antibody, the heavy chain variable region gene sequence was ligated to a knob mutation-containing human IgG1 constant region gene sequence (amino acid sequence set forth in SEQ ID NO: 30) and constructed into an EcoRI / NotI double-digested linearized pCDNA3.1 vector using homologous recombinase (purchased from Vazyme). The light chain variable region gene sequence was ligated to a human kappa light chain constant region gene sequence (amino acid sequence set forth in SEQ ID NO: 31) and constructed into an EcoRI / XhoI double-digested linearized pCDNA3.1 vector. The gene sequence encoding a hole mutation-containing human Fc region (amino acid sequence set forth in SEQ ID NO: 32) was constructed into an EcoRI / XhoI double-digested linearized pCDNA3.1 vector. This process was performed according to the manufacturer's instructions. The homologous recombination product was transferred into Top10 competent cells, and the cells were plated on ampicillin-resistant plates and cultured overnight at 37°C. Single clones were selected and sequenced, and the plasmids were extracted. Cell transfection and protein purification were performed according to Example 2.2.

[0138] Example 4: Purity measurement of anti-HER2 antibodies In this study, HPLC was used to detect the purity of the protein. The HPLC method was as follows: Mobile phase: 150 mM NaHPO·12H O, pH 7.0. Chromatography conditions: Detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.35 mL / min, detection time: 20 min, Zenix-C SEC-300 column (SEPAX 4.6 × 300 mm, 3 μm).

[0139] The results are shown in Table 2. The antibody molecules of the present invention after affinity purification had good purity and met the requirements for downstream process development.

[0140] [Table 2]

[0141] Example 5: Thermal stability of anti-HER2 monoclonal antibodies The thermal stability of various antibodies was measured by DSC (differential scanning calorimetry). Samples were concentrated and diluted to 1 mg / mL with PBS. 5000X fluorescent coloring agent Sypro Orange (purchased from Bio-Rad) was diluted 50-fold with ultrapure water to obtain 100X fluorescent coloring agent Sypro Orange. 50 μL of 1 mg / mL sample was taken, 10 μL of 100X fluorescent coloring agent Sypro Orange and 40 μL of ultrapure water were added, and mixed thoroughly. Then, 30 μL of the mixture was taken and added to a 96-well PCR plate. Three replicates for each sample were placed in the PCR machine. The heating program was set to a constant temperature of 25°C for 5 minutes, followed by heating to 99°C at a rate of 0.5°C / min. After the program was completed, the temperature value at the lowest point of the curve on the "melting curve" graph was read. This was the Tm value of the sample. The specific results are shown in Table 3 below and demonstrate that point mutations and affinity optimization do not reduce the thermostability of the antibody.

[0142] [Table 3]

[0143] Example 6: Affinity measurement of anti-HER2 antibodies ForteBio affinity measurements were performed according to existing methods (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):270-8). Briefly, the sensor was equilibrated offline in analysis buffer for 30 minutes, then detected online for 60 seconds to establish a baseline. The purified antibody obtained as described above was then added online to the AHQ sensor. The sensor was then placed in 100 nM human or cynomolgus HER2 antigen (purchased from AcroBiosystems) for 5 minutes, after which the sensor was transferred to analysis buffer and allowed to dissociate for 5 minutes. Finally, kinetic analysis was performed using a 1:1 binding model.

[0144] The experimental results are shown in Figures 2A and 2B. The affinity of the optimized anti-HER2 antibody of the present invention was equivalent to that of trastuzumab before optimization. The affinity of the trastuzumab PTM-removed single-arm antibody was significantly lower than that of the trastuzumab single-arm antibody, but the affinity of the anti-HER2 single-arm antibody of the present invention after PTM modification and affinity optimization for binding to human HER2 antigen was equal to or greater than that of the trastuzumab single-arm antibody.

[0145] Example 7: Binding of monoclonal antibodies to human HER2 In this experiment, grown and cultured N87 cells (autologous HER2 expression) were digested with 0.25% EDTA trypsin, washed once with culture medium, and then cultured at a cell density of 2 × 10 6 The diluted antibodies were adjusted to reach a concentration of 100 cells / mL and added to a 96-well flow cytometry plate at 100 μL / well. The plate was then centrifuged for further use. The gradient diluted antibodies were added to the 96-well flow cytometry plate with cells at 100 μL / well and incubated at 4°C for 60 minutes. After two washes with PBS, goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1:1000 in 2% BSA was added at 100 μL / well and incubated at 4°C for 60 minutes. After two washes with PBS, the cells were resuspended by adding 100 μL / well of PBS and detected on a CytoFlex (Beckman) flow cytometer. The corresponding mean fluorescence intensity (MFI) was calculated.

[0146] The optimized anti-HER2 antibodies of the present invention had binding activity to HER2 expressed on human gastric cancer cell line N87, which was equivalent to that of trastuzumab. Although the cell binding activity of the trastuzumab PTM-removed single-arm antibody was significantly lower than that of the trastuzumab single-arm antibody, the affinity-optimized anti-HER2 single-arm antibody had binding affinity to the HER2 antigen on N87, which was equal to or greater than that of the trastuzumab single-arm antibody and equivalent to that of a monoclonal antibody.

[0147] Example 8. Blockade of HER2 signal-dependent cell proliferation by anti-HER2 antibodies In this experiment, expanded and cultured N87 (self-expressing HER2) cells were digested with 0.25% EDTA trypsin, washed once with culture medium, and grown at a cell density of 5 × 10 4 The diluted antibodies were added to the 96-well plate at 80 μL / well with cells at 80 μL / well and incubated for 3-5 days in a cell culture incubator. Finally, cell viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) kit, and the chemiluminescent signal was collected using a microplate reader.

[0148] The results are shown in Figure 4. All of the optimized anti-HER2 antibodies of the present invention were able to significantly inhibit the proliferation of N87 cells, and this cell proliferation inhibitory effect was equivalent to that of trastuzumab monoclonal antibody.

[0149] Example 9. Induction of ADCC effect (reporter gene) by anti-HER2 antibody In this experiment, 3 × 10 cells were grown and cultured. 4 / well N87 (autologous HER2-expressing) cells and 1.2 × 10 5 NFAT-luciferase / Jurkat CD16a effector cells (overexpressing CD16a and NFAT-Luc) were mixed with 1000 cells / well and seeded into a 96-well white-bottom cell culture plate. A gradient of HER2 monoclonal antibodies was then added to the 96-well plate, mixed, and incubated for 6 hours in a cell culture incubator. The ADCC effect was detected using the Bio-Glo Luciferase Assay System (Promega, G7940) kit, and the chemiluminescence signal was then collected using a microplate reader.

[0150] The experimental results are shown in Figure 5. The optimized anti-HER2 monoclonal antibody of the present invention can mediate ADCC through HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells. In addition, the ADCC effect of the anti-HER2 (NO3-46) antibody was comparable to that of trastuzumab.

[0151] Example 10. Stability test of anti-HER2 monoclonal antibody 10.1. Sample Processing for Stability Testing of Anti-HER2 Monoclonal Antibodies The purified protein samples were diluted to 1 mg / mL with diluent, dispensed into vials, and incubated in a refrigerator at 4°C and an incubator at 40°C for 2 and 4 weeks, respectively. After the samples were treated at high temperature for 2 and 4 weeks, antibody characterization analyses and activity verification, such as charge heterogeneity analysis, PTM analysis, and activity verification, were performed.

[0152] 10.2 Charge heterogeneity analysis of anti-HER2 antibodies after high-temperature treatment Cation exchange chromatography (CEX-HPLC) was used to determine the charge heterogeneity of anti-HER2 antibody samples after high-temperature treatment. The CEX-HPLC method was as follows: Mobile phase A: 20 mM MES / MES-Na, pH 6.7; Mobile phase B: 20 ​​mM MES / MES-Na + 200 mM NaCl, pH 6.7; Chromatographic conditions: Detection wavelength: 280 nm; Column temperature: 40 °C; Flow rate: 1 mL / min; Detection time: 20 min; Gradient: 3–35 min, 0% B–100% B; Chromatographic column: ProPac WCX-10 (4 × 250 mm, 10 μm).

[0153] The experimental results are shown in Figure 6. After high-temperature accelerated treatment of trastuzumab, a significant increase in basic and acidic charge variants was observed using CEX-HPLC. Compared with trastuzumab, the basic and acidic charge variants of the anti-HER2 antibody of the present invention after PTM modification and affinity optimization did not change significantly, indicating their significantly better stability.

[0154] 10.3 PTM analysis of anti-HER2 antibodies after high-temperature treatment After high-temperature accelerated treatment, 200 μg samples of trastuzumab and modified molecules were collected and placed in 1.5 mL centrifuge tubes. 100 μL of protein denaturing solution (8 M guanidine hydrochloride, pH 6.0) and 2 μL of 1 M DTT were added, and the tubes were placed in a water bath at 37°C for 30 minutes to denature and reduce the proteins. After the water bath, immediate centrifugation was performed for 30 seconds, 4.4 μL of 1 mol / L IAM solution was added, the tubes were vortexed for 30 seconds to mix, immediately centrifuged for 30 seconds, and then left to stand in the dark for 30 minutes. After incubation in the dark, the sample was pipetted into a 10 kDa ultrafiltration tube, 300 μL of 20 mmol / L His-HCl pH 6.0 enzyme digestion buffer was added, and the tube was centrifuged at 13,000 rpm for 15 minutes to desalt the sample. The tube was then subjected to reverse centrifugation at 3,000 g for 3 minutes to recover the sample and determine the protein concentration. 40 μg of the desalted protein was taken, 2 μL of 0.5 mg / mL trypsin / Lys-C mixed enzyme solution (enzyme:protein ratio 1:40) was added, and the enzyme digestion buffer was made up to 40 μL. The tube was gently vortexed to mix, immediately centrifuged for a few seconds to prevent bubbles, and placed in a 37°C water bath for 4 hours. After enzymatic digestion, the reaction was terminated by adding 2 μL of 20% aqueous formic acid solution, mixed thoroughly, and then centrifuged at 13,000 rpm / min for 5 minutes, and the supernatant was collected and subjected to RP-UHPLC-MS analysis.

[0155] The experimental results are shown in Table 4. Trastuzumab was deamidated and isomerized at the LC-Asn-30 / HC-Asn-55 / HC-Asp-102 sites, and the deamidation and isomerization of the sample increased to a certain extent after high-temperature accelerated treatment. The results of RP-UHPLC-MS analysis showed that the degree of deamidation at the LC-Asn-30 site of trastuzumab increased significantly, while the changes in deamidation or isomerization at the HC-Asn-55 / HC-Asp-102 site were relatively small. Compared to trastuzumab, the anti-HER2 antibody of the present invention after PTM modification and affinity optimization showed no detectable deamidation or isomerization at these sites, indicating significantly better stability.

[0156] [Table 4]

[0157] 10.4 Verification of anti-HER2 antibody activity after high-temperature treatment ForteBio affinity measurements were performed according to existing methods (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):270-8). Briefly, the sensor was equilibrated offline in analysis buffer for 30 minutes, then detected online for 60 seconds to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor. The sensor was then placed in 100 nM human HER2 antigen (purchased from AcroBiosystems) for 5 minutes, after which the sensor was transferred to analysis buffer and allowed to dissociate for 5 minutes. Finally, kinetic analysis was performed using a 1:1 binding model.

[0158] The experimental results are shown in Figure 7. After 2 and 4 weeks of high-temperature treatment, the affinity of trastuzumab for binding to human HER2 antigen was significantly reduced, and the longer the high-temperature treatment time, the lower its binding activity became. Compared to trastuzumab, the anti-HER2 antibody of the present invention after PTM modification and affinity optimization showed no significant change in binding activity to HER2 antigen before and after high-temperature treatment, indicating significantly better stability.

[0159] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed, and such modifications are within the scope of protection of the present invention. The entirety of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. (1) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs): (a) a VH CDR1 having the structure represented by GFNIKDTY (SEQ ID NO: 10); (b) a VH CDR2 having the structure represented by IYPTQGYT (SEQ ID NO: 11); (c) a VH CDR3 having the structure represented by SRWGGEGFYAMDY (SEQ ID NO: 12); and / or (2) The following three light chain variable region (VL) complementarity-determining regions (CDRs): (d) X １ X ２ VQX ３ A VL CDR1 having the structure represented by: A (SEQ ID NO: 33); (e) a VL CDR2 having the structure represented by SAS (SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27); (f) QQHX ４ X ５ VL CDR3 having the structure represented by TPPT (SEQ ID NO: 34) An antibody or antigen-binding fragment thereof capable of specifically binding to HER2, comprising: During the ceremony, X １ is selected from the amino acid residues Q and N, X ２ is selected from the amino acid residues N, Y, and S; X ３ is selected from the amino acid residues G and T; X ４ is selected from the amino acid residues Y, F, and S; X ５ is selected from the amino acid residues S, M, and T; An antibody or an antigen-binding fragment thereof.

2. a VH CDR1 set forth in SEQ ID NO: 10, a VH CDR2 set forth in SEQ ID NO: 11, and a VH CDR3 set forth in SEQ ID NO: 12; a VL CDR1 set forth in any one of SEQ ID NOs: 18, 22, and 26, a VL CDR2 set forth in any one of SEQ ID NOs: 19, 23, and 27, and a VL CDR3 set forth in any one of SEQ ID NOs: 20, 24, and 28; The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. (1) VH CDR1 set forth in SEQ ID NO: 10, VH CDR2 set forth in SEQ ID NO: 11, and VH CDR3 set forth in SEQ ID NO: 12, and VL CDR1 set forth in SEQ ID NO: 18, VL CDR2 set forth in SEQ ID NO: 19, and VL CDR3 set forth in SEQ ID NO: 20, or (2) VH CDR1 set forth in SEQ ID NO: 10, VH CDR2 set forth in SEQ ID NO: 11, and VH CDR3 set forth in SEQ ID NO: 12, and VL CDR1 set forth in SEQ ID NO: 22, VL CDR2 set forth in SEQ ID NO: 23, and VL CDR3 set forth in SEQ ID NO: 24, or (3) VH CDR1 set forth in SEQ ID NO: 10, VH CDR2 set forth in SEQ ID NO: 11, and VH CDR3 set forth in SEQ ID NO: 12, and VL CDR1 set forth in SEQ ID NO: 26, VL CDR2 set forth in SEQ ID NO: 27, and VL CDR3 set forth in SEQ ID NO:

28. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

4. VH having the sequence set forth in SEQ ID NO: 9 or a variant thereof, and VL having the sequence set forth in any one of SEQ ID NOs: 17, 21, and 25 or a variant thereof. Including, 4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) or a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived, and preferably the substitutions are conservative substitutions.

5. (1) a VH having the sequence set forth in SEQ ID NO: 9 and a VL having the sequence set forth in SEQ ID NO: 17; or (2) VH having the sequence set forth in SEQ ID NO: 9 and VL having the sequence set forth in SEQ ID NO: 21, or (3) VH having the sequence set forth in SEQ ID NO: 9 and VL having the sequence set forth in SEQ ID NO: 25 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:

6. further comprising a constant region derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the light chain of the antibody or antigen-binding fragment thereof preferably comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a heavy chain constant region set forth in SEQ ID NO: 29 and / or a light chain constant region set forth in SEQ ID NO:

31.

8. The antigen-binding fragment may be Fab, Fab', (Fab') ２ 8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of an Fv, a disulfide-linked Fv, a scFv, a diabody, and a single domain antibody (sdAb).

9. (1) a first peptide chain comprising three heavy chain variable region (VH) complementarity determining regions (CDRs) defined as in any one of claims 1 to 3; (2) a second peptide chain comprising three light chain variable region (VL) complementarity determining regions (CDRs) as defined in any one of claims 1 to 3; and (3) a third peptide chain capable of forming a dimer with the first peptide chain; Including, Preferably, the first peptide chain comprises a heavy chain variable region (VH) as defined in claim 4 or 5, Preferably, a single-arm antibody, wherein the second peptide chain comprises a light chain variable region (VL) as defined in claim 4 or 5.

10. the second peptide chain further comprises a constant region derived from a human immunoglobulin; Preferably, the second peptide chain comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda); 10. The single-arm antibody of claim 9, wherein the second peptide chain preferably comprises a light chain constant region set forth in SEQ ID NO:

31.

11. The following items (1) or (2): (1) The first peptide chain further comprises a constant region derived from a human immunoglobulin, preferably the constant region derived from a human immunoglobulin is a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and preferably the heavy chain constant region has a first modification that promotes dimer formation between the first peptide chain and the third peptide chain; (2) The third peptide chain comprises an Fc domain monomer, and preferably the Fc domain monomer is an IgG Fc domain monomer, for example, an IgG1, IgG2, IgG3, or IgG4 Fc domain monomer, and preferably the Fc domain monomer has a second modification that promotes dimer formation between the third peptide chain and the first peptide chain. Further having at least one technical feature selected from Preferably, one of the first modification and the second modification is a "knob" modification and the other is a "hole" modification, thereby forming a "knob-and-hole" modification that promotes dimerization of the first peptide chain with the third peptide chain; Preferably, the first modification is a "knob" modification and the second modification is a "hole" modification, thereby forming a "knob and hole" modification that promotes dimerization of the first peptide chain with the third peptide chain; 11. The single-arm antibody of claim 9 or 10, wherein the heavy chain constant region preferably comprises the amino acid sequence set forth in SEQ ID NO: 30 and the Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO:

32.

12. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a heavy chain variable region and / or a light chain variable region thereof, or an isolated nucleic acid molecule encoding a single-arm antibody or a heavy chain variable region and / or a light chain variable region thereof according to any one of claims 9 to 11.

13. A vector comprising the nucleic acid molecule of claim 12, preferably a cloning vector or an expression vector.

14. A host cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13.

15. A method for preparing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 or a single-arm antibody described in any one of claims 9 to 11, comprising culturing a host cell described in claim 14 under conditions that allow expression of the antibody or antigen-binding fragment thereof or the single-arm antibody, and recovering the antibody or antigen-binding fragment thereof or the single-arm antibody from a culture of the cultured host cells.

16. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 or a single-arm antibody described in any one of claims 9 to 11, and optionally comprising a pharmaceutically acceptable carrier and / or excipient.

17. 15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the single-arm antibody according to any one of claims 9 to 11, the isolated nucleic acid molecule according to claim 12, the vector according to claim 13 or the host cell according to claim 14 in the manufacture of a medicament for activating HER2, increasing the activity of immune cells, enhancing an immune response, and / or preventing and / or treating a tumor or an infection in a subject, comprising: Preferably, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells; Preferably, the immune response is a HER2-mediated immune response; Preferably, the subject is a mammal, e.g., a human; Preferably, the use of said antibody or antigen-binding fragment thereof or said single-arm antibody, alone or in combination with a further pharmaceutically active agent.

18. 19. A method for enhancing an immune response and / or preventing and / or treating a tumor or an infection in a subject, comprising administering to said subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the single-arm antibody of any one of claims 9 to 11, or the pharmaceutical composition of claim 16, Preferably, the immune response is a HER2-mediated immune response; Preferably, the subject is a mammal, for example a human.

19. 12. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 or the single-arm antibody of any one of claims 9 to 11, and optionally a detectable label conjugated to said antibody or antigen-binding fragment thereof or said single-arm antibody, Preferably, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium ester compounds, luminol, and derivatives thereof or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins), radionuclides, and biotin conjugates.

20. 20. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a single-arm antibody according to any one of claims 9 to 11, or a conjugate according to claim 19, Preferably, the kit comprises a conjugate according to claim 19, Preferably, the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a second antibody capable of specifically recognizing the antibody or antigen-binding fragment thereof, and optionally the second antibody further comprises a detectable label, for example, an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin; Preferably, the kit comprises the single-arm antibody of any one of claims 9 to 11 and a second antibody capable of specifically recognizing the single-arm antibody, and optionally the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

21. 20. A method for detecting the presence or level of HER2 in a sample, comprising using an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a single-arm antibody according to any one of claims 9 to 11, or a conjugate according to claim 19, Preferably, it is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescence immunoassay, fluorescence immunoassay or radioimmunoassay; Preferably, the method comprises using a conjugate according to claim 19, Preferably, the method comprises using an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and further comprises using a second antibody bearing a detectable label for detecting the antibody or antigen-binding fragment thereof (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin), Preferably, the method comprises using the single-armed antibody of any one of claims 9 to 11, and further comprises using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol, and derivatives thereof or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the single-armed antibody.

22. 20. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a single-arm antibody according to any one of claims 9 to 11, or a conjugate according to claim 19, in the manufacture of a detection reagent for detecting the presence or level of HER2 in a sample, comprising: Preferably, the detection reagent detects the presence or level of HER2 in a sample by the method of claim 21, Preferably, the sample is a cell sample (e.g., immune cells) from a subject (e.g., a mammal, preferably a human or a cynomolgus monkey).

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